Effect of Drying

The contents of phenolic compounds in the leaves showed different fluctuations according to the drying temperature.

Secoiridoids showed a significant variation (*p* < 0.05) among samples from the same season and dried at different temperatures. Values varied from 0.6 to 24.5 μg/g, 0.1 to 7.5 μg/g, 0.6 to 4.9 μg/g, and 4.1 to 380.0 μg/g for olive leaf samples collected in January, April, August, and November, respectively. The highest amounts were registered for leaves dried at 120 ◦C followed by those dried at 80 ◦C collected in autumn, with values reaching 380 and 53 μg/g, respectively.

Oleuropein contents varied between 0.5 and 23.2 μg/g, 0.1 and 6.9 μg/g, 0.6 and 2.1 μg/g, and between 4.1 and 290.0 μg/g for olive leaf samples of January, April, August, and November, respectively. As can be seen, the highest value was registered at a drying temperature of 120 ◦C for the samples collected in November. For the two collecting times of January and April, it seems that 25 and 120 ◦C were the appropriate drying temperatures to obtain the highest secoiridoid content, while 60 and 120 ◦C leaf drying provided the highest amounts of secoiridoids in August. Finally, when collected in November, leaves dried at 80 and 120 ◦C gave the highest amounts of secoiridoids. Accordingly, we can consider a temperature of 120 ◦C as convenient to obtain higher secoiridoid contents in olive leaves.

Oleuropein content of fresh olive leaves was very low as compared to its content in dried leaves, which might be due to the cell structure destruction of drying that allows any solvent to penetrate more easily. In a previous study, it was claimed that the low area of the surface facilitates the penetration of solvents into cells [5]. On the other side, it seems that supercritical fluid extraction does not permit the extraction of oleuropein when fresh leaves are used. This is in agreemen<sup>t</sup> with the results reported in our previous work [9]. Depending on the collecting season, the appropriate drying temperature for obtaining the highest amounts of oleuropein was as follows—25 ◦C when collected in January and April, and 120 ◦C when collected in August and November, reaching 290 μg/g in the latter. Previous studies reported that the composition of olive leaf extracts is greatly influenced by the drying technique [3,7]. In other work, oleuropein content of fresh green olive leaves was very low as compared to its content in dried leaves, explained by the fact that the surface area was too low to facilitate the penetration of solvents into cells so that oleuropein stayed protected in leaves cell [5].

In the same context, secologanoside contents showed remarkable fluctuations among olive leaf samples dried at different temperatures, with the leaves dried at 120 ◦C being those that gave the highest contents in secologanoside among all samples. Its value reached 32.4 μg/g.

Elenolic acid glucoside was not detected in fresh olive leaves regardless of the sampling time. The most remarkable variation is registered for the sample dried at 120 ◦C and collected in November in which its amount reached 12.8 μg/g.

The major compound in lignans that was determined in the majority of the analyzed extracts was acetoxypinoresinol, of which amounts presented a mean value of more than 77% of determined lignans, followed by pinoresinol and syringaresinol. The highest amounts of lignans were found in fresh leaves independently of the sampling time and reached 12.6 μg/g. Their levels showed a remarkable decrease with the increase in drying temperature.

Flavonoids amounts tended to decrease when increasing the drying temperature independently of the sampling time. For samples collected in November, when increasing drying temperature, flavonoids decreased gradually but increased then at 120 ◦C. The registered values of total flavonoids

ranged from 0.2 to 0.8, 0.1 to 0.3, and 0.2 to 5.3 μg/g for samples collected in January, April, and November, respectively.

Triterpenoids showed a significant variation (*p* < 0.05) among fresh and dried olive leaf samples at different temperatures, with the highest values registered in fresh leaves of the August sampling. The contents of determined triterpenoids varied from 0.2 to 11.9 mg/g in January, between 0.2 and 0.4 mg/g in April, 0.3 and 27.4 mg/g in August, and finally from 0.7 to 18.8 mg/g in November.

### *2.3. Principal Component Analysis*

The principal component analysis permits us to better visualize the classification of the olive leaf samples under study according to the sampling time. Thus, PCA was applied to the data of the samples under study on the basis of classes of the identified phytochemicals.

As shown in Figure 5, for fresh leaves, the first two principal components (F1 and F2) explained 97.74%, 96.36%, 96.57%, 92.32%, 97.32%, 89.55% and 88.40% of the variance for fresh leaves, and leaves dried at 25, 40, 60, 80, 100, and 120 ◦C, respectively. For fresh leaves, a positive correlation was observed between the axis F1 and secoiridoids, triterpenoids, flavonoids, and simple phenolic compounds (cos<sup>2</sup> > 0.9) whereas lignans showed a positive correlation with the F2 axis. Good separation was observed between samples collected in August and the rest of the samples according to the F1 axis. Regarding the leaves dried at 25 ◦C, a positive correlation was registered between the F1 axis and secoiridoids, flavonoids, and other polar compounds (cos<sup>2</sup> > 0.85), whereas a positive correlation was observed between the F2 axis and lignans and simple phenolic compounds (cos<sup>2</sup> > 0.89). A clear separation was observed between the three main groups of samples (samples collected in January, samples collected in April, and samples collected in August and November). At a drying temperature of 40 ◦C, the PCAin Figure 5 showed a clear separation between samples collected in November and the rest of the samples. A positive correlation was registered between the F1 axis and secoiridoids, lignans, and flavonoids (cos<sup>2</sup> > 0.95). At 60 ◦C, secoiridoids, other polar compounds and lignans which correlated highly to the F1 (cos<sup>2</sup> > 0.81) axis permitted the separation between samples collected in November and the rest of samples. Three main groups of samples could be classified in the PCA applied at the data registered at 80 ◦C. Indeed, a clear separation could be visualized between samples collected in January, samples collected in November, and samples collected in April and August. When dried at 100 ◦C, lignans and triterpenoids permitted a clear separation between samples collected in April and samples collected in August, while other polar compounds correlated to the F1 axis (cos<sup>2</sup> = 0.882) and contributed to the separation of samples collected in November from the rest of samples. At 120 ◦C, secoiridoids were the most contributing variable in the classification of samples according to the F1 axis (cos<sup>2</sup> = 0.947) while simple phenolic compounds were the most contributing variable in the classification of samples according to the F2 axis (cos<sup>2</sup> = 0.851).

**Figure 5.** Principal Component Analysis of olive leaf samples according to sampling time: (**I**): Fresh leaves, (**II**): Leaves dried at 25◦C, (**III**): Leaves dried at 40 ◦C, (**IV**): Leaves dried at 60 ◦C, (**V**): Leaves dried at 80 ◦C, (**VI**): Leaves dried at 100 ◦C, and (**VII**): Leaves dried at 120 ◦C. A: January, B: April, C: August, and D: November, FL: fresh leaves.

In Figure 6, the plotting of olive leaf samples is presented according to drying temperatures. As can be observed, the most remarkable classification of samples was registered for the August and November samplings. In August, a clear separation was observed between fresh and dried leaves, regardless of the drying temperature. A high correlation was registered between the F1 axis and

lignans and flavonoids (cos<sup>2</sup> > 0.98). In November, a good separation was observed between the leaves dried at 120 ◦C and the rest of the samples. Secoiridoids and other polar compounds correlated highly to the F1 axis (cos<sup>2</sup> > 0.96).

For the majority of cases, flavonoids contents decreased with the increase of drying temperature except for November sampling time when the highest amount was registered at 120 ◦C.

**Figure 6.** Principal Component Analysis of olive leaf samples according to drying temperature. (**I**) January, (**II**) April, (**III**) August, and (**IV**) November. 1: fresh leaves, 2: 25 ◦C, 3: 40 ◦C, 4: 60 ◦C, 5: 80 ◦C, 6: 100 ◦C, and 7: 120 ◦C.

### **3. Materials and Methods**
